Method of separation by adsorption

ABSTRACT

A method of separating a selected ionic component from a sample, comprises contacting the sample with an ionic adsorbent whose charge density is such that the component is bound selectively in the absence of added ionic component that competitively binds the adsorbent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage Application of InternationalApplication No.: PCT/GB2003/003953, filed on Sep. 5, 2003, which claimspriority to GB Application No.: 0220894.0, filed on Sep. 9, 2002.

FIELD OF THE INVENTION

The present invention relates to a method of separation by adsorption.

BACKGROUND OF THE INVENTION

Under certain conditions, adsorbent materials containing ionic groupscan bind molecules of a net opposing charge. Such processes, for examplechromatography, are currently utilised in the purification andseparation of biomolecules in a complex mixture such as blood orfermentation or cell culture broths.

In column chromatography, the mixture to be analysed is applied to thetop of a column comprising an adsorbent material which acts as the“stationary phase”. A liquid solvent (the “mobile phase”) is passedthrough the column under gravity or pressure carrying the dissolvedmixture. Because the different compounds in the mixture have differentionic interactions with the mobile and stationary phases, they will becarried along in the mobile phase to varying degrees, resulting inseparation. A salt gradient is then usually applied to remove, in turn,separate bound components.

In such processes, the exact conditions for separation are typicallydetermined by trial and error. The operational selectivity of anadsorbent relates to the number of molecules that bind to it as themixture passes over; under normal conditions there is generally lowspecificity. Variation in the pH or ionic strength causes theinteraction between individual components of the mixture and adsorbentto change. The ionic strength may be varied to allow a desired componentto adsorb, but so that solvent molecules or additional componentscompete for available binding sites on the adsorbent, thus preventingthe binding of an undesired component. Selectivity also varies dependingon the physical structure of the adsorbent, for example the sizedistribution of pores or the chemical nature of the underivatisedadsorbent.

The “working capacity” (dynamic capacity) of an adsorbent refers to theamount of a particular component which will bind to and be retained onthe adsorbent. Working capacity is dependent on, inter alia, the chargedensity, ligand type and pore size distribution of the adsorbent.

Chang et al [Journal of Chromatography A, 827 (1998), 281-293] suggeststhat the protein adsorption capacity of an adsorbent is stronglycorrelated with the accessible surface area, and less so with theintrinsic adsorption affinity. The authors propose that uptake dynamicsare influenced to a large extent by mean pore size, although it isacknowledged that other structural parameters, such as pore connectivityand adsorption affinity, may also play a role.

DePhillips et al [Journal of Chromatography A, 933 (2001), 57-72]reports that, above a threshold amount, increased charge density andionic capacity do not necessarily result in increased protein retention.DePhillips et al postulate that a high charge density is relativelyunimportant, proposing that an equivalent capacity may be attained byoptimally orientating and positioning ligands of lower charge density.The data presented are based on experiments using additional salt tooptimise the ionic strength.

In summary, research in this field has, over time, suggested that anincrease in the charge density of the adsorbent leads, up to a point, toan, increased capacity, when added competing salts are used.

Analytical and production-scale chromatography differ, inter alia, inthe way in which their performance is measured. For analyticalseparations, the overriding requirement is that analysis is rapid; thisis often achieved using small, non-porous stationary phase particles.For large-scale chromatographic processes, capacity, recovery andthroughput are typically the factors on which performance is judged.Optimum performance may be achieved by trading off throughput againstselectivity and recovery. Currently, desirable working capacity isattained by using adsorbents of high charge density, with additionalsalt or buffer incorporated in the mobile phase. As mentioned above, thepresence of additional salt (e.g. NaCl) reduces the strength ofinteraction between the stationary and mobile phase, allowing thespecific binding of a particular component.

Current methodologies suffer from a number of limitations, one of whichis cost. A major factor in the manufacture of biomolecules is the costof raw materials. Buffered solutions are required to stabilisebiomolecules against variations in pH and to reduce the likelihood ofinsolubilisation (precipitation). For this reason, concentrations ofbuffer/salt components are kept to a minimum in feed solutions andusually range from 10 to 100 mM, depending on the biomolecule to bestabilised.

SUMMARY OF THE INVENTION

The present invention is based on the realisation that, rather thanusing the combination of a high charge density adsorbent and added saltin the sample, desirable working capacity may be attained by varying thecharge density of binding surfaces of the adsorbent. For example, whileDePhillips et al reports that increased charge density and ioniccapacity do not necessarily result in increased protein retention, thispublication does not, on the basis of the data presented, systematicallyaddress charge density as a retention variable.

According to the invention, a method of separating a selected ioniccomponent from a sample, comprises contacting the sample with an ionicadsorbent whose charge density is such that the component is boundselectively, in the absence of an added ionic component thatcompetitively binds the adsorbent. The charge density of the adsorbentis preferably 10 to 100 μmol/ml, more preferably 20 to 90 μmol/ml, andmost preferably 30 to 80 μmol/ml. In a preferred embodiment, theadsorbent material is cationic, the material preferably comprisingsulphopropyl groups supported on a suitable substrate, for exampleagarose or Sepharose.

The invention utilises the finding that the charge density may be lowenough to allow binding with only one ionic component, the interactionwith other components being too weak for them to bind. In this way, highselectivity and capacity may be achieved at relatively low cost, sincethe use of large quantities of additional salts is no longer necessary.

By optimising the charge density of the adsorbent surface, the inventionprovides a highly selective method of separation. A method of theinvention involves a marked reduction in the amount of adsorbent-boundcompeting molecules, allowing greater working capacities to be attainedwithin typical ranges of pH and ionic strength.

The invention bypasses the need for the inclusion of competing ionic.components (e.g. salts) in the sample. Selectivity is achieved by usingan adsorbent of predetermined charge density suitable for selectivebinding of the ionic component of interest over a range of ionicstrengths. Thus a sample may be brought directly into contact with theadsorbent, without any pretreatment.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention may be used for the separation of ionic polymericcompounds. In particular, the present invention may be used for theseparation of biomolecules such as those found in complex mixtures suchas blood and cell culture broths. A method of the invention may be usedin the production of a monoclonal or polyclonal antibody, since antibodyproduction generally requires a protein-specific purification step, suchas protein A purification.

The adsorbent used in a method of this invention is an optionallyderivatised solid phase or insoluble compound which is capable of ionicinteraction with the liquid phase. The adsorbent may comprise a ceramic,synthetic or natural polymeric material, or a mixture thereof. Forexample, the adsorbent may comprise dextran or another natural polymerchemically or physically attached to a solid phase.

Selection of an adsorbent of appropriate charge density can be achievedusing any suitable method or technique known in the art. The exact valuewill depend on factors known to those of ordinary skill in the art. Onefactor is the nature of the material to be bound and/or that whichshould not be bound. Another is the nature of the charged entities onthe adsorbent, of which a variety can be used.

Selectivity may be achieved by using an adsorbent of sufficiently lowionic charge density such that only the component of interest binds toit. An example of this is the separation of a mixture of immunoglobulin(IgG) and protein A. Sulphopropyl groups having an (cat)ionic strengthof >140 μmol/ml bind both proteins and the IgG-protein A complex.Sulphopropyl groups having weaker charge density, of approximately 75μmol/ml, are selective towards IgG, the result being that, afterelution, protein A is observed in the unbound fraction and IgG in thebound fraction.

Separation may be carried out using any suitable apparatus known in theart, for example an ion-exchange column. Elution may also be conductedby known procedures.

Techniques such as polyacrylamide gel electrophoresis (PAGE), inparticular sodium dodecyl sulphate (SDS-PAGE), may be used to analysethe various fractions of separation.

The following Examples illustrate the invention, with reference to theaccompanying drawings, in which:

FIG. 1 is a graph showing the elution profiles of three adsorbents ofdifferent charge density used in the separation of IgG and protein A.

FIG. 2 is similar to FIG. 1, except that the elution profiles pertain tothe separation of acidic cheese whey proteins.

EXAMPLE 1

Separation of Immunoglobulin and Protein A

Agarose beads were manufactured, cross-linked and chemically derivatisedwith sulphopropyl (SP) groups of varying charge densities. Forcomparison, a commercially available SP adsorbent, SP Sepharose, wasalso used.

Immunoglobulin G and protein A were mixed in the ratio 10:1 (w/w) in abuffered solution, pH 4.0-5.5 and conductivity 2-6 mSi/cm. Threedifferent SP-based cation-exchange adsorbents were analysed forselectivity of binding. These contained 75 μmol/ml SP agarose cationicgroups, 140 μmol/ml SP agarose cationic group and SP sepharosecontaining 200-250 μmol/ml cationic groups.

One column volume of the protein mixture was applied to a packed columnof buffer-equilibrated SP adsorbent at a flow rate of 100 to 300 cm/hr.The column was then washed with buffered solution to remove non-boundprotein, and a salt gradient of increasing conductivity was applied tothe column in order to elute ionically-bound proteins.

The eluent from the column was analysed for absorbance at 220 nm usingan on-line detector. A variety of pH and conductivity values wasutilised within the ranges defined above. Non-bound fractions andbound-eluted fractions were retained from each column run, and analysedfor protein content using SDS-PAGE.

The results are shown in FIG. 1. The SP adsorbents with >140 μmol/mlcationic groups bound all the protein components; IgG, protein A and thecomplex between IgG and protein. However, the adsorbent with only 75μmol/ml (dashed line) shows a substantial quantity of protein in theunbound fraction; this non-binding protein fraction was shown to beapproximately 90% protein A and 10% IgG. The eluting protein fractionwas shown to contain 98% IgG and 2% protein A. It is evident that thecationic adsorbent with lower charge density has not bound protein A.

EXAMPLE 2

Separation of Acidic Cheese Whey Proteins

Cheese whey was obtained from de-fatted milk and the pH adjusted to 4.3with dilute phosphoric acid. One column volume of the protein mixturewas applied to the three SP cationic adsorbents described in Example 1,in order to separate the different proteins. The proteins were washedfrom the column with a buffer solution of pH 4.3. The proteins wereeluted from each adsorbent column using a gradient of increasing sodiumchloride concentration (0 to 1M). The salt gradient was applied alongeach adsorbent column at an elution volume range of 12 to 50 ml. Theeluent from each column was analysed for absorbance at 280 nm using anon-line detector.

FIG. 2 shows three elution profiles (optical density at 280 nm versuselution volume) conducted using the three different cation adsorbentsunder the same binding and elution gradient conditions. The threecationic adsorbents had different ionic charge densities. The opticaldensity traces show how the acidic whey proteins were bound and elutedfrom each adsorbent.

The absorbance profiles show two proteins separated into distinct peaksfor the adsorbents having >100 μmol/ml ionic charge density. Theadsorbent with <100 μmol/ml ionic charge density was more selective,binding and eluting only one protein.

1. A method of separating immunoglobulin G, an ionic protein compound ofinterest, from a protein sample comprising protein A using a selectivecation-exchange adsorbent having a sufficiently low ionic charge densityto ionically bind to immunoglobulin G consisting essentially of thefollowing steps; (a) contacting a protein sample having a conductivitybetween 2-6 mSi/cm and containing immunoglobulin G and protein A, with aselective cation-exchange adsorbent consisting of agarose beads havingsulphopropyl groups attached thereto and having an ionic charge densityfrom 10 to 100 μmol/ml; (b) ionically binding the immunoglobulin G tothe agarose beads having sulphopropyl groups attached thereto, (c)washing the agarose beads having sulphopropyl groups attached theretowith a buffered solution to remove unbound protein A, (d) applying asalt gradient of increasing conductivity to the agarose beads havingsulphopropyl groups attached thereto, and (e) eluting the ionicallybound immunoglobulin G from the agarose beads having sulphopropyl groupsattached thereto.
 2. The method according to claim 1, wherein theselective cation-exchange adsorbent has an ionic charge density from 20to 90 μmol/ml.
 3. The method according to claim 1, wherein the selectivecation-exchange adsorbent has an ionic charge density from 30 to 80μmol/ml.
 4. The method according to claim 1, wherein the pH of proteinsample solution is 4.0-5.5.
 5. A method of separating protein A fromimmunoglobulin G in a buffered protein sample solution having a pH4:0-5.5 using a selective cation-exchange adsorbent having sulphopropylgroups, consisting essentially of the following steps: (a) contactingthe buffered protein sample solution having a pH 4.0-5.5 and aconductivity 2-6 mSi/cm, protein A and immunoglobulin G with a selectivecation-exchange adsorbent having sulphopropyl groups attached theretoand an ionic charge density from 10 to 100 μmol/ml to ionically bind tothe immunoglobulin G, and (b) washing the selective cation-exchangeadsorbent with a buffered solution to remove unbound protein A.
 6. Themethod according to claim 5, which further comprises (c) applying a saltgradient of increasing conductivity to the selective cation-exchangeadsorbent, and eluting the bound immunoglobulin G from the selectivecation-exchange adsorbent.
 7. The method according to claim 5, whereinthe selective cation-exchange adsorbent has an ionic charge density from20 to 90 μmol/ml.
 8. The method according to claim 5, wherein theselective cation-exchange adsorbent has an ionic charge density from 30to 80 μmol/ml.
 9. The method according to claim 5, wherein the selectivecation-exchange adsorbent comprises agarose beads.